glutathione
s-transferase
gst
bcl-gl
recombinant
protein
expressed
escherichia
coli
strain
bl21
codon-plus
ril
competent
cells
stratagene
purification
recombinant
proteins
performed
using
glutathione
sepharose
4b
beads
ge
healthcare
under
nondenaturing
conditions
according
supplier
instructions
confirmation
direct
binding
bcl-gl
melk
removed
gst
from
gst-fused
bcl-gl
protein
using
prescission
protease
ge
healthcare
according
supplier
instructionsrecombinant
proteins
human
appl1
n-terminal
fragments
including
bar
residues
bar-ph
domains
residues
expressed
escherichia
coli
purified
using
tag
affinity
chromatography
his-tagged
proteins
appl1
appl1
expressed
soluble
recombinant
proteins
bl21
startm
de3
strain
e.
coli
invitrogen
cells
harvested
induction
isopropyl-β-d-thiogalactopyranoside
iptg
c.
cells
lysed
lysozyme
lysate
supernatant
purified
his-selecttm
affinity
beads
sigma
both
cases
tag
removed
thrombin
recombinant
proteins
human
rab5a
variants
genbank
id
nm_004162
human
rab21
bc021901
human
rab22a
nm_020673
fused
n-terminal
gst
expressed
bl21
e.
coli
purified
gst-affinity
chromatography
sample
concentrated
mg
stored
phosphate-buffered
saline
pbs
v/v
βme
c.
recombinant
protein
human
rabaptin5
genbank
id
caa62580
expressed
purified
described
previously
zhai
et
al
two
additional
point
mutations
c719s
c734s
introduced
reduce
aggregationsoluble
fractions
filtered
syringe
filters
applied
ni-nta
affinity
column
pre-equilibrated
tris-hcl
ph
nacl
β-mercaptoethanol
target
protein
complexes
subunit
gst-tag
sv40
st
his-tag
eluted
elution
buffer
tris-hcl
ph
nacl
imidazole
β-mercaptoethanol
dialyzed
overnight
tris-hcl
ph
nacl
dtt
dialyzed
protein
applied
gst
affinity
column
remove
free
sv40
st
on-column
cleavage
tev
protease
performed
overnight
flow-through
fraction
gst
column
reapplied
ni-nta
column
remove
cleaved
his-tag
tev
protease